Modified collagen, methods of manufacture thereof

ABSTRACT

The present invention provides a modified collagen comprising S-nitroso groups and a method of manufacture of such a modified collagen. Also provided is a wound dressing comprising such a modified collagen, particularly a wound dressing comprising a formulated composition comprising such a modified collagen.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a 35 U.S.C. Section 371 national stage filingof International Patent Application No. PCT/GB2016/051025, filed 12 Apr.2016, and through which priority is claimed to United Kingdom PatentApplication GB 1506236.7, filed 13 Apr. 2015, the disclosures of whichare incorporated herein by reference in their entireties.

FIELD OF INVENTION

The present invention provides a method for the manufacture of amodified collagen molecule having one or more of vasodilative,anti-inflammatory and disinfection effects. More specifically, thepresent invention relates to a modified collagen which can be formulatedwith one or both of other collagen species and other polymers tofabricate films, membranes, hydrogels and other constructs capable ofproducing stable gel or membrane based scaffolds for application as awound healing medical device.

BACKGROUND TO THE INVENTION

Collagen is the major protein of the extracellular matrix (ECM) and isthe most abundant protein found in mammals, comprising 25% of the totalprotein content and 70% to 80% of skin (dry weight). The central featureof all collagen molecules is their stiff, triple-stranded helicalstructure. Types I, II, and III are the main types of collagen found inconnective tissue and constitute 90% of all collagen in the body.

Previously, collagens were thought to function only as a structuralsupport; however, it is now evident that collagen and collagen-derivedfragments control many cellular functions, including cell shape anddifferentiation, migration, and synthesis of a number of proteins.Findings suggest that cell contact with precise extracellular matrixmolecules influence cell behaviour by regulating the quantity andquality of matrix deposition.

Wound healing is a complex process that involves coordinatedinteractions between diverse immunological and biological systems.Long-term wounds remain a challenging clinical problem, affectingapproximately 6 million patients per year, with a high economic impact.

Wound healing is a process whereby the skin (or another organ-tissue)repairs itself after injury. In normal skin, the epidermis (outermostlayer) and dermis (inner or deeper layer) exist in a steady-stateequilibrium and shielded from the external environment. When the skin isbroken, the normal (physiologic) process of wound healing begins. Theclassic model of wound healing comprises three or four sequential, yetoverlapping, phases:

-   -   Phase 1: Haemostasis.    -   Phase 2: Inflammation.    -   Phase 3: Proliferation.    -   Phase 4: Remodelling.

Upon injury to the skin, a set of complex biochemical events takes placein a closely orchestrated cascade to repair the damage. Due to a numberof potential stimuli (local tissue ischaemia, bioburden, necrotictissue, repeated trauma, etc.), wounds can stall in the inflammatoryphase contributing to the chronicity of the wound. One key component ofchronic wounds is an elevated level of matrix metalloproteinases (MMPs).At elevated levels, MMPs not only degrade nonviable collagen but alsoviable collagen. In addition, fibroblasts in a chronic wound may notsecrete tissue inhibitors of MMPs (TIMPs) at an adequate level tocontrol the activity of MMPs. These events prevent the formation of thescaffold needed for cell migration and ultimately prevent the formationof the extracellular matrix (ECM) and granulation tissue.

Collagen-based wound dressings are uniquely suited to address the issueof elevated levels of MMPs by acting as a ‘sacrificial substrate’ in thewound. It has also been demonstrated that collagen breakdown productsare chemotactic for a variety of cell types required for the formationof granulation tissue. In addition, collagen based dressings have theability to absorb wound exudates and maintain a moist wound environment.

A number of different collagen dressings are available, which employ avariety of carriers/combining agents such as gels, pastes, polymers,oxidized regenerated cellulose (ORC), and ethylene diamine tetraaceticacid (EDTA). The collagen within these products tends to be derived frombovine, porcine, equine, or avian sources, which is purified in order torender it nonantigenic. The collagen in a given collagen dressing canvary in concentration and type. Certain collagen dressings are comprisedof Type I (native) collagen; whereas, other collagen dressings containdenatured collagen (gelatine) as well. A given collagen dressing maycontain ingredients, such as alginates and cellulose derivatives thatcan enhance absorbency, flexibility, and comfort, and help maintain amoist wound environment.

Research has shown that some collagen-based dressings produce asignificant increase in the fibroblast production; have a hydrophilicproperty that may be important in encouraging fibroblast permeation;enhance the deposition of oriented, organized collagen fibres byattracting fibroblasts and causing a directed migration of cells; aid inthe uptake and bioavailability of fibronectin; help preserve leukocytes,macrophages, fibroblasts, and epithelial cells; and assist in themaintenance of the chemical and thermostatic microenvironment of thewound. The mode of action of several collagen dressings includes theinhibition or deactivation excess MMPs. As mentioned, excess MMPs are akey contributor to wound chronicity.

Collagen dressings have a variety of pore sizes and surface areas aswell. All of these attributes are designed to enhance the woundmanagement aspects of the dressings. Many collagen dressings contain anantimicrobial agent to control pathogens within the wound. Collagendressings typically require a secondary dressing.

NO is a volatile gas produced in many tissues and organs of the body,which acts as a signalling mechanism between cells. NO exerts severaleffects including:

-   -   Vasodilation.    -   Stimulation of angiogenesis.    -   Regulation of immune responses.    -   Potent anti-microbial activity.

In recent years, NO has emerged as a critical molecule in wound healing,with NO levels increasing rapidly after skin damage and graduallydecreasing as the healing process progresses. In a study by Han G et al.titled “Nitric Oxide-Releasing Nanoparticles Accelerate Wound Healing byPromoting Fibroblast Migration and Collagen Deposition”, The AmericanJournal of Pathology. 2012; Vol. 180, No. 4, April, the authorsdemonstrated the effects of a novel NO-releasing nanoparticle technologyon wound healing in mice. The results showed that the NO-nanoparticles(NO-np) were able to significantly accelerate wound healing. It was alsofound that the NO-np was able to modify leukocyte migration and increasethe production of tumour growth factor-β in the wound area, whichsubsequently promoted angiogenesis (blood flow) to enhance the healingprocess. The authors also demonstrated that using human dermalfibroblasts, the NO-np increased fibroblast migration and collagendeposition in wounded tissue. This data shows that NO-releasingnanoparticles have the ability to modulate and accelerate wound healingin a pleiotropic manner.

While NO-nanoparticles have demonstrated that it should be feasible todeliver a localised NO effect, the ability to create collagen-NOscaffolds as a method of developing wound care devices has not beendemonstrated.

The clinical use of NO to date has been minimal, mainly due to thetechnical challenge of delivering it effectively to target tissues andalso being able to store the NO in a stable form in materials.Additional technical challenges of NO include being able to release themolecule from a medical device at the correct rate (kinetics) for itsparticular use.

In diabetic foot ulcer wounds, a combination of the associated chronicmicrobial infection and inflammation can lead to blood capillarydegradation (reduced blood flow) and amputation. It is desirable for amedical device targeting diabetic foot ulcers (DFU) to:

-   -   Provide clinical benefits in reducing the associated infection.    -   Provide the ability to stimulate blood flow to the affected        region.    -   Promote wound healing.    -   Meet the growing market demand for safer more efficacious        products for treatment of DFU.

The present invention seeks to meet the problems outlined above throughdevelopment of a S-nitroso collagen to deliver exogenous NO,particularly a S-nitroso collagen based scaffold, such as a S-nitrosocollagen I, II, III, IV, V, VI, IX X or XI based scaffold derived fromeither mammalian or marine sources and more preferably from jellyfish.The benefits of this technology are the production of a localised andcontrolled release of nitric oxide (NO) to help stimulate blood flow andalso the treatment of microbial infection associated with DFU. Currenttherapeutic strategies have proved suboptimal in the treatment of DFUand it is imperative to focus on new therapeutic approaches and thedevelopment of technologies for both short and long-term woundmanagement.

This invention provides NO release systems for wound care involving thesynthesis of modified collagen comprising S-nitroso groups.

S-Nitrosothiols, also called thionitrites, may serve as carriers in themechanism of action of endothelium relaxing factor (EDRF) by stabilisingthe labile NO radical from inactivation by reactive oxygen species andthus delivering Nitric Oxide to the site of wound damage causingincreased blood flow and effective treatment of the microbial infection.

The ability to supply exogenous NO is advantageous because it restoresthe body's natural defence and signalling system.

SUMMARY OF INVENTION

In a first aspect of the invention, there is provided a method ofproducing a modified collagen, comprising at least the steps of:

-   -   providing a collagen comprising a S—S bond;    -   introducing a —SH group in said collagen comprising a S—S bond        by reduction of the S—S bond to provide a collagen thiol        comprising a —SH group;    -   nitrosating the —SH group of the collagen thiol to provide a        modified collagen, said modified collagen comprising S-nitroso        groups.

In one embodiment, the collagen is preferably collagen of one or more ofType I, II, III, IV, V, VI, IX, X and XI. In another embodiment, thecollagen is a collagen other than collagen of one or more of Type I, II,III, IV, V, VI, IX, X and XI, such as collagen of one or more of TypeVII, VIII, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, XX, XXI, XXII,XXIII, XXIV, XXV, XXVI, XXVII, XXVIII and XXIX.

In one embodiment, the collagen is preferably one or more of Type I, IIand V. In another embodiment, the collagen is preferably one or more ofType IV and XI like materials.

In one embodiment, the step of providing a collagen comprising a S—Sbond, comprises at least the steps of:

-   -   providing a source collagen comprising one or both of lysine and        hydroxylysine residues;    -   reacting the source collagen with an activated dicarboxylic acid        derivative comprising a disulphide group to form amide bonds        between the carbonyl function of the activated dicarboxylic acid        derivative and the ε-NH₂ groups of one or both of the lysine and        hydroxylysine residues, thereby providing the collagen        comprising a S—S bond.

In another embodiment, the source collagen is one or more of Type I, II,III, IV, V, VI, IX, X and XI, said source collagen comprising one orboth of lysine and hydroxylysine residues and the collagen comprisingthe S—S bond is one or more of Type I, II, III, IV, V, VI, IX, X and XIcomprising a S—S bond.

In another embodiment, the source collagen is selected from one or bothof pepsin solubilised collagen and acid solubilised collagen, preferablypepsin solubilised collagen.

In another embodiment, the activated dicarboxylic acid derivative is acompound of the formula:ZN—C(O)—R³—C(O)—NH—R¹—S—S—R²—NH—C(O)—R³—C(O)—NZ  (I)

-   -   wherein R¹, R² and R³ independently represent divalent linking        groups; and    -   ZN together represent a nitrogen containing heterocyclic group.

In another embodiment, the activated dicarboxylic acid derivativecomprises the compound:

In another embodiment, the step of providing a collagen comprising a S—Sbond, comprises at least the steps of:

-   -   providing a collagen-binding protein wherein the        collagen-binding protein comprises a cysteine residue comprising        —SH groups;    -   reacting the cysteine residue —SH groups of the collagen-binding        protein with a photoreactive cross-linker to provide a        photoreactive cross-linker modified collagen-binding protein        comprising a S—S group;    -   combining the photoreactive cross-linker modified        collagen-binding protein with a collagen to provide a complex of        the photoreactive cross-linker modified collagen-binding protein        and the collagen;    -   irradiating the complex with electromagnetic radiation to        cross-link the photoreactive cross-linker with the collagen to        provide the collagen comprising a S—S bond.

In another embodiment, the collagen comprising the S—S bond is one ormore of Type I, II, III, IV, V, VI, IX, X and XI comprising a S—S bond.

In another embodiment, the step of providing the collagen-bindingprotein comprising a cysteine residue is carried out by site-directedmutagenesis of a collagen-binding protein.

In another embodiment, the photoreactive cross-linker isN-[4-(p-azidosalicylamido)butyl]-3′-(2′pyridyldithio) propionamide(APDP) and the electromagnetic radiation is ultraviolet radiation.

In another embodiment, the S-nitroso groups are in the vicinity of aprotein binding site on the modified collagen.

In another embodiment, the step of introducing a —SH group in saidcollagen comprising a S—S bond by reduction of the S—S bond to provide acollagen thiol having a —SH group comprises:

-   -   reacting the collagen comprising a S—S bond with dithiothreitol.

In another embodiment, the nitrosating of the —SH group comprises one ormore of:

-   -   reacting the —SH group of the collagen thiol with a solution of        acidified nitrite;    -   reacting the —SH group of the collagen thiol with gaseous NO;        and    -   reacting the —SH group of the collagen thiol with a        S-nitrosothiol.

In another embodiment, the nitrosating of the —SH group is carried outin the presence of a chelating agent, such as EDTA.

In another embodiment, the nitrosating of the —SH group is carried belowroom temperature, such as a temperature of less than 5° C., preferably atemperature of less than 0° C.

In another embodiment, the method further comprises the step of:

-   -   incorporating the modified collagen into a one or more of the        group comprising a type II collagen, a type V collagen and a        gelatin matrix; or    -   incorporating the modified collagen into a polymer, preferably a        film-forming polymer, a membrane-forming polymer, a        hydrogel-forming polymer or a polymer that is able to fabricate        films, membranes, hydrogels and other constructs capable of        producing stable gel or membrane based scaffolds.

In another embodiment, the collagen, particularly a collagen of one ormore of Type I, II, III, IV, V, VI, IX, X and XI, is derived frommammalian or non-mammalian sources, such as natural or GMO sources.

In another embodiment, the non-mammalian source is one or more selectedfrom jellyfish, marine invertebrate and fish.

In another embodiment, the collagen, such as the collagen derived from anon-mammalian source, is atelocollagen.

In a second aspect, there is provided a modified collagen obtainable bythe method of the first aspect or its embodiments.

In a third aspect, there is provided a modified collagen comprisingS-nitroso groups.

In one embodiment, the modified collagen is preferably derived fromcollagen of one or more of Type I, II, III, IV, V, VI, IX, X and X. Inanother embodiment, the collagen is a collagen other than collagen ofone or more of Type I, II, III, IV, V, VI, IX, X and XI, such ascollagen of one or more of Type VII, VIII, XII, XIII, XIV, XV, XVI,XVII, XVIII, XIX, XX, XXI, XXII, XXIII, XXIV, XXV, XXVI, XXVII, XXVIIIand XXIX.

In one embodiment, the modified collagen is derived from mammalian ornon-mammalian sources, such as natural or GMO sources.

In another embodiment, the non-mammalian source from which the modifiedcollagen is derived is one or more selected from jellyfish, marineinvertebrate and fish.

In another embodiment, the modified collagen is derived fromatelocollagen. The atelocollagen may be produced by pepsin enzymedigestion of a source collagen.

In another embodiment, the modified collagen comprising S-nitroso groupscomprises a lysine or hydroxylysine residue of formula (IX):

-   -   in which R¹ and R³ are as defined above, and preferably        independently represent ethanediyl or propanediyl groups, most        preferably ethanediyl i.e. —CH₂CH₂—;    -   R⁵ is H or OH;    -   X is selected from the group OH or a chemical bond; and Y is        selected from H or a chemical bond, with the proviso that one or        both of X and Y are chemical bonds forming peptide bonds within        the modified collagen.

In a fourth aspect, the modified collagen is for use as a medicament.

In one embodiment, the modified collagen is for use in wound healing.

In another embodiment, the wound healing is the treatment of a wound,preferably an ulcer, more preferably a diabetic ulcer, particularly adiabetic foot ulcer.

In a fifth aspect, there is provided a wound dressing comprising aformulated composition comprising the modified collagen incorporatedinto one or more of the group comprising a type II collagen derived froma jellyfish, a type V collagen derived from a jellyfish and a gelatinmatrix.

In a sixth aspect, there is provided a wound dressing comprising aformulated composition comprising the modified collagen incorporatedinto a polymer based matrix scaffold comprising of either chondroitin,hyaluronic acid, silicon, or some other polymer based technologyapplicable for wound application.

In one embodiment of the wound dressing, the formulated composition is afilm, membrane or hydrogel composite.

In another embodiment of the wound dressing, said film, membrane orhydrogel composite has a thickness in a range of from 5 to 200micrometer.

In another embodiment of the wound dressing, said film, membrane orhydrogel composite is sterilised, preferably during formation of thewound dressing.

In another embodiment of the wound dressing, said film, membrane orhydrogel composite has a porous structure.

In another embodiment of the wound dressing, said collagen derived froma jellyfish comprises Rhizostoma pulmo jellyfish tissue.

In another embodiment of the wound dressing, it further comprises foodgrade agents.

In another embodiment of the wound dressing, it further comprisesmedical grade agents.

In another embodiment of the wound dressing, it has enhancedvasodilation effects, particularly with respect to blood flow control.

In another embodiment of the wound dressing, it acts as a disinfectionagent.

In another embodiment of the wound dressing, it has antimicrobialproperties, particularly against bacteria, yeasts, fungi and viruses.

In a seventh aspect, there is provided a method of S-nitrosation of the—SH-containing groups within collagen thiol comprising one or more ofexposure to nitric oxide (NO) delivered from acidified nitrite, exposureto NO gas and reaction with a S-nitrosothiol.

In one embodiment, the modified collagen is treated with an excess of aS-nitrosothiol.

In another embodiment, the S-nitrosothiol is one or more selected fromthe group comprising S-nitroso-glutathione,S-nitroso-N-acetylpenicillamine and S-nitroso-cysteine.

FIGURES

Embodiments of the present invention will now be described, by way ofexample only, with reference to the accompanying figures in which:

FIG. 1a ) shows the structure of a photoreactive cross-linker,N-[4-(p-azidosalicylamido)butyl]-3′-(2′pyridyldithio) propionamide(APDP), which can be used in the method disclosed herein.

FIG. 1b ) shows a scheme for one method of introducing sulfhydryl groupsinto a collagen as disclosed herein.

FIG. 2 shows the absorption spectrum in the wavelength range of 290 to365 nm of a modified collagen comprising a S—H group after 10 minutestreatment with a nitrosating agent together with a control sample towhich no nitrosating agent was added, as detailed in the Example below.

DETAILED EXAMPLES OF THE INVENTION

In the following detailed description, numerous specific details are setforth in order to provide a thorough understanding of the presentinvention. It will be understood by those of ordinary skill in the artthat embodiments of the present invention may be practiced without thesespecific details while still remaining within the scope of the claims.

Type I collagen is the most abundant structural component of the dermalmatrix and migrating keratinocytes likely interact with this protein.Collagenase (via the formation of gelatin) may aid in dissociatingkeratinocytes from collagen-rich matrix and thereby promote efficientmigration over the dermal and provisional matrices.

Cellular functions are regulated by the extracellular matrix (ECM). Theinformation provided by ECM macromolecules is processed and transducedinto the cells by specialized cell surface receptors. Evidencedemonstrates that the receptors play a major function in contraction ofwounds, migration of epithelial cells, collagen deposition, andinduction of matrix-degrading collagenase. Although keratinocytes willadhere to denatured collagen (gelatin), collagenase production is notturned on in response to this substrate. Keratinocytes have been knownto recognize and migrate on Type I collagen substratum, resulting inenhanced collagenase production. Collagen therefore plays a key role ineach phase of wound healing.

The present invention relates to a method of producing a modifiedcollagen comprising S-nitroso groups. Sulfhydryl (—SH) groups inproteins represent an abundant source of reduced thiol for interactionwith NO and S-nitroso-proteins which form readily under physiologicalconditions. The major limitation to determining protein-binding sites oncollagen has been a lack of useful methods for introducing suchsulfhydryl groups.

The modified collagen described herein may be produced from collagen ofone or more of Type I, II, III, IV, V, IX and X, also referred to hereinas “source” collagen. The modified collagen may be produced fromcollagen derived from mammalian or non-mammalian sources. Preferably,when the collagen is derived from mammalian sources, it is derived fromnon-human mammalian sources. The non-mammalian source may be one or moresource selected from jellyfish, marine invertebrate and fish. Themodified collagen may be produced from collagen derived from natural orgenetically modified organism (GMO) sources.

The first step of the method described herein comprises providing acollagen in which the collagen comprises a S—S bond. Preferably thecollagen is one or more of Type I, II, III, IV, V, VI, IX, X and XI.Alternatively the collagen is collagen other than one or more of Type I,II, III, IV, V, VI, IX, X and XI, such as collagen of one or more ofType VII, VIII, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, XX, XXI,XXII, XXIII, XXIV, XXV, XXVI, XXVII, XXVIII and XXIX.

Naturally occurring collagen does not typically comprise a S—S bond.Disulphide groups may be incorporated into collagen by a number ofroutes. In a preferred embodiment, the collagen comprising a S—S bond isobtainable by providing a source collagen, in which the collagencomprises one or both of lysine and hydroxylysine residues. Forinstance, the source collagen may be collagen of one or more of Type I,II, III, IV, V, VI, IX, X and XI having one or both of lysine andhydroxylysin residues.

Lysine and hydroxylysine are α-amino acids having ε-amino groups. Asused herein, the term “residue” refers to the portion of a chemicalcompound remaining after incorporation into, for instance by chemicalreaction and bond formation, another substance. Thus, amino acid“residue” refers to the polymerised form of an amino acid monomerpresent in a polypeptide. Collagen is formed of a triple-helix ofpolypeptide chains, one or more of which commonly comprise one or bothof lysine and hydroxylysine residues. Lysine is present as one of theten most abundant amino acid residues in both mammalian and fishcollagen. Although 4-5 times less plentiful in collagen than lysineresidue, hydroxylysine residue is also present in amounts sufficient tobe of use in the method disclosed herein. Thus, a collagen comprising alysine or hydroxylysine residue, prior to treatment as described herein,may comprise a residue of formula (XI):

-   -   in which R⁵ is H or OH. When R⁵ is H a lysine residue is        present. When R⁵ is OH a hydroxylysine residue is present; and    -   X is selected from the group OH and a chemical bond and Y is        selected from H and a chemical bond, with the proviso that one        or both of X and Y are chemical bonds forming peptide bonds        within collagen. The peptide chain forming part of the modified        collagen is shown bracketed by “[ ]” in formula (XI). The        residue could be in a terminal position of the peptide, for        instance if one or other of X and Y is OH and H respectively. If        both X and Y are peptide bonds, the lysine residue is        non-terminal within the peptide chain forming part of the        collagen.

The source collagen comprising one or both of lysine and hydroxylysineresidues is preferably solubilised source collagen comprising one orboth of lysine and hydroxylysine. The solubilisation may be achieved bypepsin digestion or acid digestion to provide pepsin solubilised sourcecollagen comprising one or both of lysine and hydroxylysine or by aciddigestion to provide acid solubilised source collagen comprising one orboth of lysine and hydroxylysine.

The source collagen, such as a pepsin solubilised or acid solubilisedsource collagen, comprising one or both of lysine and hydroxylysineresidues, can be reacted with an activated dicarboxylic acid derivativecomprising a disulphide (i.e. S—S) group to provide collagen comprisinga S—S bond, such as collagen of one or more of Type I, II, III, IV, V,VI, IX, X and XI comprising a S—S bond. In this reaction, the carbonylgroup of the activated dicarboxylic acid derivative can react with theε-amino group of the lysine or hydroxylysine residues present in thecollagen to form an amide bond.

The activated dicarboxylic acid derivative is preferably a compound ofthe formula:ZN—C(O)—R³—C(O)—NH—R¹—S—S—R²—NH—C(O)—R³—C(O)—NZ  (I)

-   -   wherein R¹, R² and R³ independently represent divalent linking        groups, preferably divalent organic linking groups, more        preferably divalent hydrocarbon linking groups, such as an        alkanediyl group having from 1 to 6 carbon atoms or alkendiyl or        alkyndiyl groups having from 2 to 6 carbon atoms. Still more        preferably, R¹, R² and R³ independently represent ethanediyl or        propanediyl groups, most preferably ethanediyl i.e. —CH₂CH₂—.        The groups R¹, R² and R³ may independently be optionally        substituted by replacing one to four hydrogen atoms with a        hydroxyl group or a halogen, such as F or Cl. In a preferred        embodiment, R¹ and R² are identical; and    -   ZN together represent a nitrogen containing heterocyclic group,        preferably a nitrogen containing heterocyclic group having 5-6        atoms in the heterocyclic ring, in which the N atom is directly        bonded to the carbonyl group of the compound of formula (I) such        that Z represents a divalent linking group in which the two        valences are bonded to the nitrogen. The heterocyclic group may        be saturated or unsaturated, such that Z may represent an        alkanediyl, alkendiyl or alkyndiyl, particularly having 2 to 4        carbon atoms. Z may optionally comprise 1 or 2 heteroatoms        selected from O, S and N.    -   More preferably, ZN is a nitrogen containing heteroaryl group        having 5-6 atoms in the aryl ring of which from 1-3 atoms are        heteroatoms selected from O, N and S at least 1 of which is N        which is directly bonded to the carbonyl group of the compound        of formula (I). Still more preferably the heteroaryl group has        5-6 atoms in the aryl ring of which 2 atoms are N.        Alternatively, ZN is a nitrogen containing heterocyclic group        having 5-6 atoms in the heterocyclic ring having 1 N atom which        is directly bonded to the carbonyl group of the compound of        formula (I) and Z is a α, ω-organodionediyl. For example, the α,        ω-organodionediyl may be represented as —C(O)R⁴C(O)— in which R⁴        is an alkanediyl or alkendiyl having from 2 or 3 carbon atoms.    -   The group ZN may be optionally substituted by replacing from one        to four hydrogen atoms with a hydroxyl group or a halogen, such        as F, Br or Cl or by replacing two hydrogen atoms bonded to the        same carbon with an oxygen atom to form a carbonyl group,        wherein the latter substitution may occur once or twice.    -   Most preferably, the group ZN is:

Preferred activated dicarboxylic acid derivatives may be selected from:

The activated dicarboxylic acid derivative (I) may be synthesised in twosteps. Firstly, a diamino disulphide of formula (IV) may be reacted withat least two molar equivalents of a dicarboxylic acid anhydride offormula (V) to provide a dicarboxylic acid diamide of formula (VI):

-   -   in which R¹, R² and R³ are as defined above. It will be apparent        that when R¹ and R² are identical, the diamino disulphide of        formula (IV) is a symmetrical molecule, which will result in a        symmetrical activated dicarboxylic acid derivative (I).

The first reaction step may be carried out by dissolving the diaminodisulphide of formula (IV) in a solvent, such as water, and adding thedicarboxylic acid anhydride of formula (V). It is preferred that thereaction is carried out under basic conditions, such that prior to theaddition of the acid anhydride, a base can be added. For instance,aqueous sodium hydroxide can be added to adjust the pH to 10. Afteraddition of the dicarboxylic acid anhydride, the pH may decrease, and itis preferred to maintain the pH in the range of from 7 to 10 during thereaction by the addition of further base. The reaction may be carriedout at room temperature under stirring and may be complete within 30minutes to 2 hours. The dicarboxylic acid diamide product of formula(VI) may be precipitated by lowering the pH, for instance to a pH of 1,by the addition of acid, such as aqueous hydrochloric acid. Theprecipitated dicarboxylic acid diamide (VI) can be isolated byfiltration, washed with water and then dried under reduced pressure.

In the second step of the synthesis the dicarboxylic acid diamide (VI)is activated by the addition of a nitrogen containing heterocycliccompound to provide the activated dicarboxylic acid derivative (I):HO—C(O)—R³—C(O)—NH—R¹—S—S—R²—NH—C(O)—R³—C(O)—OH(VI)→ZN—C(O)—R³—C(O)—NH—R¹—S—S—R²—NH—C(O)—R³—C(O)—NZ  (I)

-   -   in which R¹, R², R³ and NZ are as defined above.

In one embodiment, the nitrogen containing heterocyclic compound may bea carbodiimide, such as a compound of formula (VII):

-   -   in which Z is as defined above. Preferably, ZN together are a        nitrogen containing heteroaryl group having 5-6 atoms in the        aryl ring of which from 1-3 atoms are heteroatoms selected from        O, N and S at least 1 of which is N. The carbodiimide (VII) is        more preferably 1,1′-carbonyl-diimidazole or the like.

At least 2 molar equivalents of the carbodiimide should be used per moleof dicarboxylic acid diamide (VI). Theoretically, the reaction willproduce 2 molar equivalents of carbon dioxide and 2 molar equivalents ofimidazole, per mole of dicarboxylic acid diamide (VI). The evolution ofcarbon dioxide gas indicates that the reaction is proceeding. Thereaction may be carried out under reduced pressure.

In another embodiment, the nitrogen containing heterocyclic compound maybe a N-hydroxy heterocyclic compound of formula (VIII):

-   -   in which ZN together is a nitrogen containing heterocyclic group        having 5-6 atoms in the heterocyclic ring of which 1 is N and Z        is a α, ω-organodionediyl. For example, the α, ω-organodionediyl        may be represented as —C(O)R⁴C(O)— in which R⁴ is an alkanediyl        or alkendiyl having from 2 or 3 carbon atoms. The N-hydroxy        heterocyclic compound (VIII) is most preferably N-hydroxy        succinimide.

The second reaction step may be carried out by dissolving thedicarboxylic acid diamide (VI) in a solvent, such as anhydrousdimethylformamide and then adding the nitrogen containing heterocycliccompound (VII) or (VIII). The activated dicarboxylic acid derivative (I)precipitates from the solution. The product can be collected byfiltration, washed with anhydrous ethyl acetate, and dried under reducedpressure.

Returning to the first step of the method of the invention, a sourcecollagen comprising one or both of lysine and hydroxylysine residues canbe reacted with an activated dicarboxylic acid derivative comprising adisulphide group, such as the activated dicarboxylic acid derivative offormula (I) to provide collagen comprising a S—S bond, such as collagenof one or more of Type I, II, III, IV, V, VI, IX, X and XI comprising aS—S bond. In this reaction, a carbonyl group of the activateddicarboxylic acid derivative can react with the ε-amino group of thelysine or hydroxylysine residues present in the collagen to form anamide bond, thereby incorporating the disulphide group. The reaction canbe represented by:ZN—C(O)—R³—C(O)—NH—R¹—S—S—R²—NH—C(O)—R³—C(O)—NZ(I)+collagen-NH₂→ZN—C(O)—R³—C(O)—NH—R¹—S—S—R²—NH—C(O)—R³—C(O)—NH-collagen+HNZ

This reaction may continue to provide a cross-linked collagen when bothactivated carboxyl groups of the activated dicarboxylic acid derivativereact with collagen, particularly different collagen triple helices orfibrils:ZN—C(O)—R³—C(O)—NH—R¹—S—S—R²—NH—C(O)—R³—C(O)—NH-collagen+collagen-NH₂→Collagen-HN—C(O)—R³—C(O)—NH—R¹—S—S—R²—NH—C(O)—R³—C(O)—NH-collagen+HNZ

The reaction can be carried out by dissolving the source collagen in asolvent. The dissolution of the source collagen can be carried out in atwo-step process. In the first step, the source collagen may be mixedwith methanol. In the second step, a polar aprotic solvent is added. Forinstance, the source collagen can be added to a mixture of methanol anddimethylsulfoxide, and allowed to swell. Additional dimethylsulfoxidecan be added with stirring until dissolution of the source collagen iscomplete. Methanol can then be removed from the solution by evaporatingunder reduced pressure. This solubilising process can be used with bothatelocollagen and telocollagen.

The activated dicarboxylic acid derivative can then be dissolved in asolvent, particularly an anhydrous polar aprotic solvent, such asdimethylsulfoxide. Since the activated dicarboxylic acid derivative offormula (I) is sensitive to water, the reaction with collagen ispreferably carried out in anhydrous polar aprotic solvents, such asdimethylsulfoxide. The activated dicarboxylic acid derivative dissolvedin a solvent is then added to the source collagen solution. The carbonylgroup of the activated dicarboxylic acid derivative can react with theε-amino group of the lysine or hydroxylysine residues present in thesource collagen to form an amide bond.

The mixture can be stirred at room temperature, for instance 22° C.,until a gel is formed. The mixture containing the gel can then be leftundisturbed e.g. for 12-18 hr. The dimethylsulfoxide can then beextracted from the gel by blending with an excess of acetone, collectingthe collagen gel by decantation and then reblending with more acetone.The mixture can then be stirred e.g. for 0.5 to 1 hr and the collagensubsequently isolated by filtration, washed with acetone, then washedwith water-ethanol (30:70 v/v) and dehydrated with ethanol. A collagencomprising a S—S bond is thereby provided, such as a collagen of one ormore of Type I, II, III, IV, V, IX and X comprising a S—S bond.

In an alternative embodiment, the collagen comprising a S—S bond may beprovided by modifying a collagen-binding protein to include aphotoreactive cross-linker comprising a disulphide group, combining thiswith source collagen to provide a complex and irradiating the complex tocross-link the photoreactive cross-linker to incorporate the disulphidegroup into the collagen. For instance, the collagen comprising a S—Sbond may be collagen of one or more of Type I, II, III, IV, V, VI, IX, Xand XI comprising a S—S bond and may be produced from source collagen ofone or more of Type I, II, III, IV, V, VI, IX, X and XI.

This route creates protein-binding sites on collagen by using asite-specific photo-cross-linking strategy allowing the creation ofthiol groups in collagen. This involves the introduction of a cysteineresidue into the collagen-binding protein by site-directed mutagenesis.A photoreactive cross-linker, preferably APDP, can be introduced intocysteine —SH groups on proteins. The complex of APDP-modified proteinand collagen can be cross-linked by ultraviolet (uv) irradiation. Thedisulfide cross-link can then be cleaved by reduction, and an —SH groupis generated on collagen allowing for subsequent S-nitrosation.

In a first step, a collagen-binding protein comprising a cysteineresidue is provided, as shown in FIG. 1b ), step a. A cysteine residueis necessary because this comprises a sulphydryl group necessary forreaction with the cross-linker. The collagen-binding protein may be, forinstance, pigment epithelium-derived factor (PEDF). PEDF is a knownanti-angiogenic/neurotrophic factor with a collagen binding siteidentified by Yasui et al as disclosed in Biochemistry, 2003, 42, pages3160-3167.

If necessary, cysteine may be incorporated into the collagen-bindingprotein if not already present or if the collagen-binding protein doesnot contain sufficient cysteine. Cysteine substitutions can be made viasite-directed mutagenesis, for instance where the collagen binding siteis localised (F383) and on the opposite surface of the site (Y211).Methods for carrying out such site-directed mutagenesis are found in J.D. J. Biol. Chem. 2002, 277, 4223-4231 and R. R. Biochemistry 1992, 31,9526-9532.

The sulphydryl groups introduced as a result of the cysteinesubstitutions can then be reacted with a photoreactive cross-linker asshown in FIG. 1b ), step b. The photoreactive cross-linker should bebifunctional. In particular, the photoreactive cross-linker shouldcomprise a functional group capable of reacting with a sulphydryl groupto produce a disulphide bond. One such suitable functional group is apyridyl-dithio group i.e. C₅NH₅—S—S—, particularly 2-pyridyl dithio. Thephotoreactive cross-linker should also comprise a functional groupcapable of cross-linking with collagen under photo-irradiation. One suchsuitable functional group is an azide group, particularly an aryl azidegroup, such as a phenyl azide, especially para —C₆H₄—N₃.

N-[4-(p-azidosalicylamido)butyl]-3′-(2′pyridyldithio) propionamide(APDP) is one example of a preferred photoreactive cross-linker and itsstructure is shown in FIG. 1a ). The disulphide group of APDP can reactwith the sulphydryl group of the cysteine to produce a disulphide bondbetween the cysteine and the photoreactive cross-linker, therebyproviding a photoreactive cross-linker modified collagen binding proteincomprising a S—S group. 2-pyridyl thione is liberated as part of thisreaction as a leaving group.

The photoreactive cross-linker modified collagen-binding protein canthen be combined with a collagen, such as collagen of one or more ofType I, II, III, IV, V, VI, IX, X and XI, to provide a complex of thephotoreactive cross-linker modified collagen-binding protein and thecollagen, as shown in FIG. 1b ), step c. In this step, the photoreactivecross-linker modified collagen-binding protein binds to the proteinbinding site of the collagen to form the complex. The complex of thephotoreactive cross-linker modified collagen-binding protein and thecollagen can then be irradiated, for example with uv light. Irradiationcauses the functional group capable of cross-linking present on thephotoreactive cross-linker to form a covalent bond with the adjacentcollagen in the complex, as shown in FIG. 1b ), step d. For example,when the functional group capable of cross-linking is a phenyl azide,irradiation at a wavelength in the range of from 250 to 280 nm willgenerate a nitrene, which can then attack nucleophilic or activehydrogen groups, such as C—H or C—NH₂ on the collagen to generate across-link by insertion across the C—H or N—H bond. In this way, thephotoreactive cross-linker modified collagen binding protein comprisinga S—S group is incorporated into the collagen to provide collagencomprising a S—S bond. It will be apparent that the disulphide groupwill be provided in the vicinity of the collagen protein binding site bythis route.

A sulphydryl group can then be introduced into the collagen comprising aS—S bond which can be provided by either of the methods discussed abovei.e. using the activated dicarboxylic acid derivate or photoreactivecross-linker methods. The collagen comprising a S—S bond can be reactedwith a suitable reducing agent, for instance as shown in FIG. 1b ), stepe. The reducing agent reduces the disulphide bond to two sulphydrylgroups, thereby cleaving the activated dicarboxylic acid derivativeresidue or the photoreactive cross-linker residue, in which thedisulphide group is located. Such a reduction proceeds by two sequentialthiol-disulfide exchange reactions, resulting in the reduction of thedisulphide group to produce collagen comprising a sulphydryl (—SH)group.

Suitable reducing agents include, for example, dithiothreitol (DTT),(2S)-2-amino-1,4-dimercaptobutane (DTBA) and tris(2-carboxyethyl)phosphine HCl (TCEP hydrochloride). Dithiothreitol is a preferredreducing agent.

The reduction step can be carried out by adding the collagen comprisinga S—S bond to a buffer solution, such as a glycine/sodium hydroxidebuffer solution at a pH of in the range of from 7.5 to 9.5, morepreferably about 8 to 9.5, preferably about 8.0. The collagen comprisinga S—S bond may be inherently acidic, and if so, neutralisation with abase, such as sodium hydroxide, may be required.

Preferably, at least two molar equivalents of DTT reducing agent can beadded per mole of disulphide group in the same buffer and the reactionallowed to proceed at 30° C. for 2-6 hr. After completion of thereaction, the pH of the liquid may be decreased to 2, for instance usingHCl. The mixture may then be dialysed with dilute HCl solution,centrifuged and freeze-dried to provide the collagen thiol having a —SHgroup.

The reduction may cause a slight degradation of the collagen chains.Consequently, shorter reaction times, lower pH and lower temperature canall be used to minimise any degradation.

It will be apparent that when the collagen comprising a S—S bond isprovided using a photoreactive cross-linker modified collagen-bindingprotein, the modified collagen-binding protein will still be attached tothe collagen via the photoreactive cross-linker as shown in FIG. 1b ),step d prior to the step of forming the collagen thiol. Reduction of theS—S bond will cleave the S—S bond of the photoreactive cross-linker.

When the source collagen comprises one or both of lysine andhydroxylysine residues, a collagen thiol comprising a lysine orhydroxylysine residue of formula (X) is provided:

-   -   in which R¹, R³ and R⁵ are as defined above. For instance, R¹        and R³ are independently selected from divalent linking groups,        preferably divalent organic linking groups, more preferably        divalent hydrocarbon linking groups, such as an alkanediyl group        having from 1 to 6 carbon atoms or alkendiyl or alkyndiyl groups        having from 2 to 6 carbon atoms. Still more preferably, R¹ and        R³ independently represent ethanediyl or propanediyl groups,        most preferably ethanediyl i.e. —CH₂CH₂—. R⁵ is H when the amino        acid residue is a lysine residue. R⁵ is OH when the amino acid        residue is a hydroxylysine residue;    -   X is selected from the group OH and a chemical bond and Y is        selected from H and a chemical bond, with the proviso that one        or both of X and Y are chemical bonds forming peptide bonds        within collagen. The peptide chain forming part of the modified        collagen is shown bracketed by “[ ]” in formula (X). The residue        could be in a terminal position of the peptide, for instance if        one or other of X and Y is OH and H respectively. If both X and        Y are peptide bonds, the lysine residue is non-terminal within        the peptide chain forming part of the collagen.

The collagen thiol having a —SH group can then be nitrosated to providea modified collagen comprising S-nitroso groups, as shown in FIG. 1b ),step f. The S-nitrosation may be carried out by any technique known inthe art for the S-nitrosation of proteins. It is preferred that thenitrosation is carried out in the presence of a chelating agent, such asEDTA, in order to improve the stability of the collagen comprising anS-nitroso group product, for instance by eliminating the presence oftransition metal ions, which catalyse the decomposition of the S-nitrosogroup. It is further preferred that the nitrosation is carried out atbelow room temperature, such as a temperature of less than 5° C., morepreferably at a temperature of less than 0° C., in order to minimise anydegradation of the S-nitroso product.

For instance, the collagen thiol may be reacted with an aqueous solutionof acidified nitrite. The nitrite may be an alkali metal nitrite, suchas sodium or potassium nitrite, preferably sodium nitrite. Theacidification can be carried out using a buffer, such as phosphatebuffered saline. As the nitrosation reaction progresses, hydrogen ionsare liberated, leading to an increase in the acidity of the solutionwhich is stabilised by the buffer. Dilute solutions of nitrite salts,when acidified can produce free nitrous acid which react with asulphydryl group to form a S-nitroso group on the collagen and yieldwater as a byproduct. Such a reaction is disclosed by Byler D M, GosserD K, Susi H in a 1983 paper titled ‘Spectroscopic Estimation of theExtent of S-Nitrosothiol Formation by Nitrite Action onSulfhydryl-Groups’ in Journal of Agricultural and Food Chemistry 31:523-527. Wink D A, Nims R W, Darbyshire J F, et al. in 1994 investigated‘Reaction-Kinetics for Nitrosation of Cysteine and Glutathione inAerobic Nitric-Oxide Solutions at Neutral pH—Insights into the Fate andPhysiological-Effects of Intermediates Generated in the NO/O-2 Reaction’in Chemical Research in Toxicology 7: 519-525.

Alternatively, the collagen thiol may be reacted with gaseous NO. Thismay be achieved my bubbling gaseous NO through the collagen thiol in asolvent. The collagen thiol may also be reacted with a S-nitrosothiol.Preferably, the S-nitrosothiol is a relatively labile and of lowmolecular weight, such as a gaseous compound. More preferably, theS-nitrosothiol is one or more compounds selected from the groupcomprising S-nitroso-glutathione, S-nitroso-N-acetylpenicillamine andS-nitroso-cysteine. Reaction with a S-nitrosothiol may be carried out ina dialysis bag and low molecular weight reagents and products dialyzedout to leave S-nitrosated collagen.

The nitrosation reaction can be monitored spectrophotometrically becauseof the existence of S—NO specific absorption peaks in the range of330-340 nm, such as at 339 nm and in the range of 540-600 nm, such as at545 nm in the uv-visible spectrum. The absorption peaks can be monitoredin the presence and absence of Cu⁺ ions, known to release NO fromS-nitrosothiols. Both the generation of NO (by NO electrode) and thereduction in absorbance at a wavelength in the range of from 330-340 nm,such as λ=339 nm is indicative of successful generation of NO releasefrom the S-nitroso collagen.

Similarly, specific absorption peaks in the range of 1480-1530 cm−1 areindicative of the stretching vibration of the N═O bond of aS-nitrosothiol in the infrared spectrum. A second absorption peak in therange of 600-730 cm⁻¹ is characteristic of the vibration of the C—S bondin the S-nitrosothiol. When the source collagen comprises one or both oflysine and hydroxylysine residues, a modified collagen comprising alysine or hydroxylysine residue of formula (IX) is provided:

-   -   in which R¹, R³ and R⁵ are as defined above. For instance, R¹        and R³ are independently selected from divalent linking groups,        preferably divalent organic linking groups, more preferably        divalent hydrocarbon linking groups, such as an alkanediyl group        having from 1 to 6 carbon atoms or alkendiyl or alkyndiyl groups        having from 2 to 6 carbon atoms. Still more preferably, R¹ and        R³ independently represent ethanediyl or propanediyl groups,        most preferably ethanediyl i.e. —CH₂CH₂—. R⁵ is H when the amino        acid residue is a lysine residue. R⁵ is OH when the amino acid        residue is a hydroxylysine residue;    -   X is selected from the group OH and a chemical bond and Y is        selected from H and a chemical bond, with the proviso that one        or both of X and Y are chemical bonds forming peptide bonds        within collagen. The peptide chain forming part of the modified        collagen is shown bracketed by “[ ]” in formula (X). The residue        could be in a terminal position of the peptide, for instance if        one or other of X and Y is OH and H respectively. If both X and        Y are peptide bonds, the lysine residue is non-terminal within        the peptide chain forming part of the modified collagen.

When the modified collagen comprises a lysine or hydroxylysine residuecomprising a S-nitroso group, such as those discussed above, forinstance modified collagen comprising a residue of formula (IX),improved stability of the S-nitroso group to decomposition is observed.For instance, modified collagen comprising a lysine or hydroxylysineresidue comprising a S-nitroso group exhibits an approximately 10-foldincrease in stability compared to cysteine residues comprising aS-nitroso group.

The modified collagen comprising a S-nitroso group product may bepurified, for instance by dialysis. It is preferred that the modifiedcollagen is dialysed against a phosphate buffer comprising ethylenediamine tetraacetic acid (EDTA) in water.

In another embodiment, the modified collagen described herein may beincorporated into a one or more of the group comprising a type IIcollagen, a type V collagen and a gelatin matrix. Preferably, thecollagen is derived from a jellyfish, such as Rhizostoma pulmo jellyfishtissue i.e. Barrel jellyfish.

The modified collagen described herein may be for use as a medicament.

In one embodiment, the modified collagen is for use in wound healing.Thus, also disclosed herein is a method of treatment of a wound in anindividual, comprising at least the step of attaching a modifiedcollagen as described herein to the wound. The modified collagen may beattached by any conventional means, such as binding e.g. as part of awound dressing, gluing, suturing etc. The wound may be an ulcer, such asa diabetic ulcer, for instance a diabetic foot ulcer.

Formulation of the modified collagen for maximum delivery of localisedand controlled release of NO to support beneficial clinical effects canbe either delivered in a gel matrix (e.g. extracellular matrix) form ormore preferably formulated into any suitable polymer material scaffoldmade or composed of one or more of collagen, gelatine, hyaluronic acid,polymethylcellulose, alginate, alginic acid and silicone basedmaterials.

There is also provided a wound dressing comprising a formulatedcomposition comprising the modified collagen described hereinincorporated into one or more of the group comprising a type II collagenderived from a jellyfish, a type V collagen derived from a jellyfish anda gelatin matrix.

Preferably, when the collagen is derived from a jellyfish it is derivedfrom Rhizostoma pulmo jellyfish tissue i.e. Barrel jellyfish.

The formulated composition of the wound dressing may be a film, membraneor hydrogel composite. Preferably, the wound dressing has a thickness ina range of from 5 to 200 micrometers. As used herein the term‘thickness’ refers to the smallest dimension of the wound dressing.

The wound dressing may further comprise food grade agents.

The wound dressing may further comprise medical grade agents.

It is preferred that the film, membrane or hydrogel composite issterilised, preferably during formation of the wound dressing. Thesterilisation may be achieved using filter sterilisation, such as with afilter in the range of from 0.22 μm to 0.45 μm or by treatment withEthylene Oxide (EtO) or by gamma irradiation.

The wound dressing is preferably stored below room temperature,preferably at a temperature below 4° C. The wound dressing is preferablystored under anhydrous conditions. The wound dressing is preferablystored under an inert atmosphere, such as under nitrogen. These stepsminimise decomposition of the S-nitroso groups.

The wound dressing disclosed herein provides enhanced vasodilationeffects with respect to blood flow control due to the presence of theS-nitroso groups of the modified collagen. The S-nitroso groups canrelease nitric oxide under physiological conditions. Nitric oxide isknown to provide vasodilation effects.

In another embodiment, the wound dressing acts as an effectivedisinfection agent. Endogenously produced nitric oxide is an importantcomponent of the body's natural defence mechanism. Depending on itsconcentration, NO exerts antimicrobial effects in two ways. At lowconcentrations, NO acts as a signalling molecule that promotes thegrowth and activity of immune cells. At high concentrations, such asduring the respiratory burst of a neutrophil, NO covalently binds DNA,proteins and lipids, thereby inhibiting or killing target pathogens.Considering that NO is an integral and highly conserved part of the hostimmune response, it is not surprising that few bacteria are able toescape the antimicrobial effect of NO.

In another embodiment, the wound dressing is antimicrobial. Inparticular, the wound dressing exhibits antimicrobial properties againstbacteria, yeasts, fungi and viruses. This is because the nitric oxidereleased by the S-nitroso groups of the modified collagen exertsantimicrobial activity.

Various further aspects and embodiments of the present invention will beapparent to those skilled in the art in view of the present disclosure.

Other aspects and embodiments of the invention provide the aspects andembodiments described above with the term “comprising” replaced by theterm “consisting of” and the aspects and embodiments described abovewith the term “comprising” replaced by the term “consisting essentiallyof”.

EXPERIMENTAL

The following experimental example describes the synthesis of a modifiedcollagen comprising S-nitroso groups from a jellyfish source. Thehalf-life of the modified collagen comprising S-nitroso groups is alsoinvestigated.

The collagen source was obtained from a barrel jellyfish, Rhizostomapulmo. A 1.003 g sample of jellyfish collagen (comprising collagenderived from mesogloea and oral arms) were first enzyme digested byaddition of from 0.1% to 0.001% v/v pepsin (Sigma) to provideatelocollagen. Firstly, a pepsin enzyme stock solution of 10 mg/ml wasmade in 0.1 M acetic acid and mixed on a vortex then stored at 4° C.This enzyme stock solution was added until a final concentration of from0.1% to 0.001% v/v was reached, more preferably a final concentration of0.01% v/v pepsin. All enzyme collagen digest samples were stored at 4°C.

The atelocollagen was then swelled in 200-400 mL methanol (RathburnChemicals Ltd.). The swollen atelocollagen did not dissolve afterstirring for 1 hr.

In order to dissolve the swollen atelocollagen, 40 mL dimethylsulfoxide(Rathburn Chemicals Ltd.) was added and the mixture stirred for 20minutes with 1 minute sonication. A further 200-450 mL dimethylsulfoxidewas added and the mixture stirred for 1 hr with intermittent sonicationfor periods of 30 s to provide an atelocollagen solution indimethylsulfoxide.

The methanol was removed from the atelocollagen solution under reducedpressure at a temperature of 35° C.

A 400 mg sample of N,N′-disuccinoyl cystamine dissolved in 16 mLdimethylsulfoxide was added to the atelocollagen solution, which wasthen covered and stirred 2.5 hrs.

The mixture was then allowed to stand for 16 hrs after which a yellowliquid with a strong odour was obtained. There was no change to theviscosity.

The atelocollagen comprising an S—S bond was then blended with acetone(Brenntag) in a ratio of 2:1 v/v acetone:atelocollagen mixture. Anoff-white, flaky precipitate was formed after 30 seconds on the firstblend. The blend was left to stand for 1 hr at a temperature of <10° C.,preferably at a temperature of 2-5° C., after which the settling of someprecipitate was observed, with the rest of the particles remaining insuspension.

The atelocollagen comprising an S—S bond was collected by centrifugationand removal of supernatant. The blending with acetone, settling andcentrifugation was repeated two further times in order to completeprecipitation.

The atelocollagen comprising an S—S bond precipitate was then filteredand washed with acetone. The precipitate collected by gravitationalfiltration using glass fibre filter paper with a pore size of 1.6 μm.The precipitate was washed five times with 100 mL acetone to yield athick yellow-brown gel-like material.

The atelocollagen comprising an S—S bond precipitate was then rinsedwith 100 mL 70:30 v/v denatured ethanol (Sigma)/water which resulted inthe precipitate coalescing together to form an elastic-like, grey-brownmaterial.

After the denatured ethanol wash, the coalesced precipitate wasfreeze-dried to provide 375 mg of an off-which chalk-like atelocollagencomprising an S—S bond.

The freeze-dried atelocollagen comprising an S—S bond was mixed with 50mL of 0.1 M glycine/NaOH buffer having a pH of 9.5. After vortexing andvigorous shaking a yellow-brown gel-like material was formed.

To this collagen mixture, of dithiothreitol (Sigma) was added and the pHfell to 8.04. The atelocollagen completely dissolved after stirring for4 hr to yield a more viscous, pale peach solution of atelocollagencomprising an S—H group (compound of formula (X) in which R¹ and R³ are—CH₂CH₂— and R⁵ is H).

The atelocollagen comprising an S—H group was then dialysed with 18 mMHCl (Fischer Scientific). The dialysis was repeated once with freshdialysis medium. There was no change in the consistency of the liquidafter 24.5 hrs.

The dialysed atelocollagen comprising an S—H group was then centrifugedto provide a pellet of a solid modified collagen and a pale yellowfibrous supernatant.

A 343 mg sample of the atelocollagen comprising an S—H group was thenadded to 5 mL phosphate buffered saline (Fischer Scientific) to form agelatinous ball having a total weight of 5.34 g. A 0.4313 g sample ofthe gelatinous ball was cut and chopped into a glass universal vial,then dissolved in 2 ml chelex treated H₂O in a water bath at 37° C. Thesolution had a pH of 2.19.

Prior to nitrosation, 40 μM EDTA (Sigma) was added.

The sample was then split into 2 aliquots, the first kept at roomtemperature, and the second chilled with ice.

A 0.003 g sample of 40 mM sodium nitrite (Sigma) was added to eachaliquot to initiate nitrosation process. The mixtures were wrapped foilto eliminate light. Both aliquots became visibly rose pink within 2.5minutes.

Aliquot a) was examined on Variskan plate reader over a range wavelengthof 290 to 450 nm at 5, 10, 20, 30, 60 and 90 minutes. FIG. 2 shows theabsorption spectrum for the sample at 10 minutes. A control plate towhich no sodium nitrite had been added was also examined and thespectrum is also shown in FIG. 2. It is apparent that the spectrum forthe nitrite treated sample at 10 minutes shows a significant absorptionpeak at around 335 nm, indicative of S-nitroso formation.

Aliquot b) on ice became extremely viscous and so samples could not bepipetted into a well plate. The colour remained vivid however and at 60minutes the sample was warmed in the water bath to return it to itsfluid state again.

It is to be understood that the application discloses all combinationsof any of the above aspects and embodiments described above with eachother, unless the context demands otherwise. Similarly, the applicationdiscloses all combinations of the preferred and/or optional featureseither singly or together with any of the other aspects, unless thecontext demands otherwise.

The invention claimed is:
 1. A modified collagen comprising S-nitrosogroups obtainable by the method comprising at least the steps of:providing a collagen comprising a S—S bond, wherein the source of thecollagen is one or more selected from the group consisting of jellyfish,marine invertebrates, and fish; introducing a —SH group in said collagencomprising a S—S bond by reduction of the S—S bond to provide a collagenthiol comprising a SH group; nitrosating the SH group of the collagenthiol to provide a modified collagen, said modified collagen comprisingone or both of lysine and hydroxylysine residues comprising S-nitrosogroups, wherein the one or both of the lysine and hydroxylysine residuesis represented by the formula (IX):

in which R¹ and R³ are independently divalent linking groups; R⁵ is H orOH; X is the group OH or a chemical bond; and Y is H or a chemical bond,with the proviso that one or both of X and Y are chemical bonds formingpeptide bonds within the modified collagen.
 2. The modified collagenaccording to claim 1 for use as a medicament.
 3. The modified collagenaccording to claim 1 for use in wound healing.
 4. A wound dressingcomprising a formulated composition comprising the modified collagenaccording to claim 1 and a gelatin matrix, wherein the collagen is oneor more selected from the group consisting of a type I collagen derivedfrom a jellyfish, a type II collagen derived from a jellyfish, and atype V collagen derived from a jellyfish.
 5. The wound dressing of claim4, wherein said collagen derived from a jellyfish comprises Rhizostomapulmo jellyfish tissue.
 6. The wound dressing of claim 4, furthercomprising food grade agents or medical grade agents.
 7. The wounddressing of claim 4 for use as a vasodilation agent or for use as adisinfection agent.
 8. The wound dressing of claim 7 havingantimicrobial properties against bacteria, yeasts, fungi and viruses. 9.A wound dressing comprising a formulated composition comprising themodified collagen according to claim 1 incorporated into any polymermatrix.
 10. The wound dressing of claim 9 where said formulatedcomposition is a film, membrane or hydrogel composite.
 11. The wounddressing of claim 10 wherein said film, membrane or hydrogel compositehas a thickness in a range of from 5 to 1000 micrometer.
 12. The wounddressing of claim 10, wherein said film, membrane or hydrogel compositeis sterilized.
 13. The wound dressing of claim 10, wherein said film,membrane or hydrogel composite has a porous structure.
 14. The modifiedcollagen of claim 1, wherein the source of collagen comprises one ormore of Type I, II, III, IV, V, VI, IX, X and XI collagen comprising aS—S bond.
 15. A modified collagen comprising one or both of lysine andhydroxylysine residues comprising S-nitroso groups, wherein the sourceof the collagen is one or more selected from the group consisting ofjellyfish, marine invertebrates, and fish, wherein the one or both ofthe lysine and hydroxylysine residues is represented by the formula(IX):

in which R¹ and R³ are independently divalent linking groups; R⁵ is H orOH; X is the group OH or a chemical bond; and Y is H or a chemical bond,with the proviso that one or both of X and Y are chemical bonds formingpeptide bonds within the modified collagen.
 16. The modified collagen ofclaim 15, wherein the collagen comprises one or more of Type I, II, III,IV, V, VI, IX, X and XI.